Adhesive composition for joining fungus spores to the surface of vegetable seeds in a stable manner and method for quantifying the adhesion capacity

ABSTRACT

An adhesive composition for joining fungus spores to the surface of vegetable seeds in a stable manner, characterized in that it includes an aqueous solution of fermented maize liquor and/or soya protein hydrolysates. A method and to the use of the same for quantifying the adhesion capacity by means of an indirect measurement in which fluorescent microspheres joined to the surface of seeds are quantified by means of the fluorescence microscopy technique is also disclosed. This method allows an indirect assessment of the adhesion of fungus spores to the surface of seeds in a simple manner and consequently allows the quality of the adhesive composition of the present invention to be assessed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the U.S. national phase of PCT Application No.PCT/ES2016/070735 filed on Oct. 19, 2016, which claims priority to ESPatent Application No. P201531523 filed on Oct. 22, 2015, thedisclosures of which are incorporated in their entirety by referenceherein.

TECHNICAL FIELD

The present invention belongs to the branch of agricultural sciences andspecifically relates to an adhesive composition for joining fungusspores to the surface of vegetable seeds in a stable manner as well asto the method for quantifying the adhesion capacity by means of anindirect measurement in which fluorescent microspheres joined to theseed surface are quantified by means of fluorescence microscopytechnology. This method enables an indirect assessment of the adhesionof fungus spores to the seed surface in a simple manner and consequentlyallows the quality of the adhesive composition of the present inventionto be assessed. Another object of the present invention is the use ofthe adhesive composition of the present invention to catalyze theadhesion of fungus spores to the surface of vegetable seeds.

BACKGROUND

Mycorrhizal association is a nutritional strategy which many plants andsome fungi have developed which ensures them a mutual benefit. Themycorrhizae are organs formed by the root of a plant and the mycelium ofa fungus. They act as an absorption system which extends through thesoil, thus providing a symbiotic relationship that is beneficial bothfor the plant and for the fungus. In terms of the plant, the growththereof is facilitated (since the fungus allows the water and therequired minerals to be absorbed better, stimulating the growth of theroot), improvement in the absorption of phosphate and other nutrients,it is better protected from the toxic effects caused by highconcentrations of determined minerals, it resists the lack of waterbetter, it is better protected against the attack of pathogens, therooting and growth of seedlings is stimulated, it helps to overcomeenvironmental stress situations. In addition, the fungus receives sugarsoriginating from photosynthesis from the plant, essentially starch.

Approximately 90% of all plant species live in symbiosis with a largequantity of fungi in the soil. This form of living in community orsymbiosis (which symbolizes the mutual benefit between the plant speciesand the fungi) is termed mycorrhiza. Since it is such an extensivephenomenon, the term “mycorrhizae” has become the name with which fungiinvolved in the formation thereof have been designated, although suchdesignation is not very orthodox. The extension of the term andcolloquial habits have led to the coining of terms such as “mycorrhize”,that is to say, placing the mycorrhizal fungi into contact with plantsor the term “mycorrhization” to indicate the establishment of thesymbiosis.

To this end, it is known from the state of the art to use mycorrhizalfungi to provide the plants with the previously mentioned benefits aswell as to use pesticide compositions, and for them to facilitate theactivity of microorganisms.

Among the documents which have been found in the closest state of theart, there is the Russian patent RU2113794C1, which describes a methodfor preparing a microbial pesticide with bipolar cover. The describedmethod comprises mixing a biopolymer with water, heating the mixturefrom 80 to 121° C. up to gel or paste formation, cooling to thetemperature from room point to 60° C. The mixture comprises one of thefollowing microorganism strains: Bacillus subtilis krietiensis ATCC55078 or 55079, Pseudomonas fluorescens ATCC 27663, Gliocladium vixensATCC 52045, Trichoderma reesei ATCC28217, Trichoderma harzianum ATCC52445, Trichoderma hamatum ATCC 52198, Trichoderma viride ATCC 52440,Streptomyces cacaoi asoensis ATCC 19093, Bacillus thuringiensis ATCC13367, Beauveria bassiana ATCC 26851, 48585 or 48023, Hirsutellathomsonii ATCC 24874, Metarhizium flavoviride ATCC 32969, Verticilliumlencanii ATCC 46578, Collectotrichum gloeosporioides f. sp. jussiaeasATCC 52634. The mixture obtained is formed and dried and a pesticide isprepared. Prior to mixing the cooled gel or paste (biopolymer) with themicroorganisms or in the process of stabilizing the mixture, thenutrients are expediently added. The following substances may be used asbiopolymers (one component), grains, tubers, root crops, starch, wax andnatural resins and the derivatives thereof. The stabilizing agents:glycerol, milk, milk.

In addition, the international patent application WO9107869A1 describesa continuous coating for seeds which comprises a liquid formulation incombination with a microorganism to biologically control plant pathogensand a solid material in carbon particle form which includes soft coal,shales, moss, muck soil, activated charcoal or mixtures which protectthe seed and the plant from soil-borne pathogens including Pythium spp.The compositions comprise the bioprotective strain Trichoderma 22 (ATCCn° 20847) which is particularly effective at protecting a variety ofcrops such as tomato, cucumber, cabbage, sugar beet, beetroot, maizefield, sweetcorn, cotton, bean, green bean and soya. The compositioncomprises an adhesive which is within the group composed of polyvinylalcohol, polyalkyleneoxide, polyvinyl acetate, starch, cellulose esterssuch as methyl cellulose and hydroxypropyl cellulose, organic silicates,including organic silicon esters, rubbers, alginate and similar.

In order to have the correct interaction between the fungi and theplants, in particular in order to achieve the positive effect of thesame in stimulating and germinating the seeds, effective adhesion of thespores to the surface of the same is required which may vary dependingon the species of plants and fungi. To this end, it is of greatimportance to develop a strategy which allows the joining of spores toseeds to be obtained in an effective and stable manner as well as to beable to quantitatively assess the efficacy of said strategy for carryingout anticipated effect monitoring.

Among the different known methods for carrying out monitoring of ananticipated effect, one of the most important methods is electronicmicroscopy quantification using fluorescent molecular markers that areeasily traceable.

Therefore, various methods of microscopy quantification by means of theuse of a wide range of fluorescent molecular markers are known in thestate of the art. For example, DsRed and GFP (green fluorescent protein)have been used for marking Rhizophagus intraradices using a biolisticapproach.

Another example is the transformation of Agrobacterium tumefaciens whichhas been recently proposed for “in vivo” monitoring of arbuscularmycorrhizal fungi during symbiotic development (Helber and Requena,2008, New Phytologist, 177: 537 to 548). However, the authors of thispublication reported that the expression of fluorescence in transformedspores was transitory and disappeared very quickly. In addition, theefficacy of the transformation mediated by Agrobacterium of spores ofthe arbuscular mycorrhizal species was very low.

However, to date, no adhesive composition has been described whichcatalyzes the joining of fungus spores to the surface of vegetable seedsto achieve a stable and effective joining which guarantees the symbiosisphenomenon.

As a result, a method has also not been described in the state of theart which allows the joining of said fungus spores to the surface ofseeds to be systematically quantified and monitored by means offluorescence microscopy in order to prove the efficacy of said joining.Therefore it is necessary to provide a simple and effective method whichallows the adhesion of the spores of the fungi to the seeds to bequantitatively evaluated in order to determine the effectiveness of theadhesion method.

The present invention thus relates to an adhesive composition forjoining fungus spores to the surface of vegetable seeds in a stablemanner which comprises an aqueous solution of fermented maize liquorand/or soya protein hydrolysates.

The present invention also relates to the method for obtaining saidadhesive composition as well as to the use of the composition object ofthe present invention for the adhesion of fungus spores to the surfaceof vegetable seeds.

The present invention also relates to an indirect method which allowsthe efficacy of the adhesive composition to be quantitatively assessedcharacterized in that silica or polyethylene microspheres are used thatare fluorescent when they are exposed to ultraviolet (UV) light withsimilar characteristics to the spores in terms of the size distribution,the spherical shape, density, specific surface and affinity for surfacewater, owing to which the effectiveness of an adhesive compositionintended for adhering fungus spores to vegetable seeds may bedetermined.

As a result, the present invention provides an adhesive composition offungus spores to vegetable seeds for promoting or catalyzing thesymbiosis phenomenon and the method for verifying or quantifying saidadhesion which allows the quality of the adhesive composition to beassessed.

The method is very advantageous since it allows an indirect measurementof the adhesion phenomenon of fungus spores to be obtained, inparticular arbuscular mycorrhizae to vegetable seeds which arepreferably cereal seeds.

Another advantage of the present invention, in addition to avoiding theuse of organisms, in particular genetically modified organisms, is thatthe fluorescent microspheres present fluorescence in the ultraviolet(UV) spectrum which is a much more stable fluorescence than ifarbuscular mycorrhizal spores were used which express fluorescentmarkers.

The present invention has an important practical application since itprovides an adhesive composition for joining fungus spores to thesurface of vegetable seeds in a stable manner which comprises an aqueoussolution of fermented maize liquor and/or soya protein hydrolysates andit provides a simple and reliable quantitative method for determiningthe effectiveness of said adhesive composition in the adhesion offluorescent microspheres to the vegetable seeds and based on the resultobtained the effectiveness of said composition for the adhesion offungus spores, with similar characteristics to the fluorescentmicrospheres, can be inferred.

SUMMARY

The present invention relates to an adhesive composition for joiningfungus spores to the surface of vegetable seeds in a stable manner whichcomprises an aqueous solution of fermented maize liquor and/or soyaprotein hydrolysates.

For the object of the present invention, “join in a stable manner” isunderstood as any action or putting a method into practice which allowsa material joining (fungus spores) to the surface of a medium (vegetableseeds) selected from the group formed by: adsorption method, covalentbond method, cross-linking and auto-immobilization method, method forjoining by means of hydrogen bridges and method for van der Waals forcebonding.

The present invention also relates to the method for obtaining saidadhesive composition as well as to the use of the composition object ofthe present invention for the adhesion of fungus spores to the surfaceof vegetable seeds.

The present invention also relates to an indirect method which allowsthe adhesion efficacy or capacity of the adhesive composition to bequantitatively assessed characterized in that silica or polyethylenemicrospheres are used that are fluorescent when they are exposed toultraviolet (UV) light with similar characteristics to the spores interms of the size distribution, the spherical shape, density, specificsurface and affinity for surface water, owing to which the effectivenessof an adhesive composition intended for adhering fungus spores tovegetable seeds may be determined.

As a result, the present invention provides an adhesive composition offungus spores to vegetable seeds for promoting or catalyzing thesymbiosis phenomenon and the method for verifying or quantifying saidadhesion which allows the quality of the adhesive composition to beassessed.

The method is very advantageous since it allows an indirect measurementof the adhesion phenomenon of fungus spores to be obtained, inparticular arbuscular mycorrhizae to vegetable seeds which arepreferably cereal seeds.

The present invention has an important practical application since itprovides an adhesive composition for joining fungus spores to thesurface of vegetable seeds in a stable manner which comprises an aqueoussolution of fermented maize liquor and/or soya protein hydrolysates andit provides a simple and reliable quantitative method for determiningthe effectiveness of said adhesive composition in the adhesion offluorescent microspheres to the vegetable seeds and based on the resultobtained the effectiveness of said composition for the adhesion offungus spores, with similar characteristics to the fluorescentmicrospheres, can be inferred.

The present invention also proposes a novel method for indirectlyquantifying the adhesion capacity of fungus spores to the surface ofseeds, particularly arbuscular mycorrhizal seeds and is based on the useof an adhesive composition which comprises protein carbohydrates and/orprotein hydrolysates to which fluorescent microspheres are added.

The peculiarity of the fluorescent microspheres which are used in thepresent invention is that they are produced from silica, beingtransparent when they are exposed to daylight, while they emitfluorescence with a yellow-green color when they are exposed toultraviolet (UV) light corresponding to a wavelength of 365 nm. Saidfluorescent spheres are not object of the present invention, but ratherthey are obtained on the market and are considered known by a personskilled in the art.

The method is based on the fact that the microspheres that arefluorescent to UV light have similar characteristics to the spores ofthe mycorrhizae, in terms of size distribution, spherical shape,density, specific surface and their affinity for surface water.Specifically, the microspheres used in the present invention have adensity of 0.98 g/cm³ and a particle size range of 1-120 microns.

The mycorrhizae are preferably arbuscular mycorrhizae Rhizophagusintraradices and Funneliformis mosseae.

The adhesive composition comprises between 1 and 10 g of fermented maizeliquor and/or 3 to 30 ml of soya protein hydrolysate in a volume ofbetween 50 and 100 ml of water.

The adhesive composition is preferably a liquid composition which isprepared by means of method of simple mixing and continuous stirring of1 and 10 g of fermented maize liquor and/or 3 to 30 ml of soya proteinhydrolysate in a volume of between 50 and 100 ml of water todissolution.

In a preferred manner, but not limiting the invention, the method forpreparing the adhesive composition comprises the following steps:

a. -) taking between 1 and 10 g of fermented maize liquor and/or 5 to 30ml of soya protein hydrolysate,

b. -) mixing with a volume of 50 ml of water,

c. -) stirring to complete homogenization, and

d. -) Adding sterile water whilst stirring to a final volume of 100 mlof final adhesive composition, obtaining the adhesive composition objectof the present invention.

The method for indirectly quantifying the adhesion capacity of fungusspores to the surface of vegetable seeds by means of an adhesivecomposition and fluorescent microspheres of the present inventioncomprises the following steps:

a. -) taking the adhesive composition of the present invention,

b. -) mixing the adhesive composition of step a) with silica orpolyethylene fluorescent microspheres with a particle size of between1-120 microns and continuing to stir,

c. -) placing the vegetable seeds into contact with the mixture of stepb),

d. -) drying the seeds at room temperature,

e. -) obtaining images of the fluorescent microspheres adhered to theseeds by fluorescence microscopy, exposing them to ultraviolet (UV)light corresponding to a wavelength of 365 nm, and

f. -) counting the fluorescent microspheres adhered to the seeds basedon the images obtained in step e).

According to a preferred aspect, step c) is implemented by submergingthe vegetable seeds in the mixture of step b) in a receptacle in avolumetric ratio (seeds:final volume) of 1:3, continuing to stir themixture for at least 1 minute between 50 and 100 rpm, concluding withdecantation and removing the excess liquid from the seeds.

According to another preferred aspect, step c) is carried out byspraying the mixture of step b) on the vegetable seeds in a homogenousmanner using a proportion of 1 l of mixture per 100 kg of seeds.

The adhesive composition of the present invention is preferably, but notlimited to, an aqueous solution which comprises soya protein hydrolysateand/or fermented maize liquor.

The quantities of the components of the present adhesive compositionaccording to the preferred embodiment are the following: soya proteinhydrolysate between 5 to 30 ml and water to complete a final volume of100 ml; fermented maize liquor between 1 to 10 g and water to complete afinal volume 100 ml or soya protein hydrolysate between 3 to 30 ml,fermented maize liquor between 1 to 10 g and water to complete a finalvolume of 100 ml.

An adhesive composition for joining fungus spores to the surface ofvegetable seeds is an object of the invention which comprises an aqueoussolution of fermented maize liquor and/or soya protein hydrolysates.

According to a significant aspect, the adhesive composition comprisesfrom 3 to 30 ml of soya protein hydrolysate and/or 1 to 10 g offermented maize liquor in water to complete a final volume of 100 ml.

Another object of the present invention is a method for preparing thepreviously cited adhesive composition which comprises the followingsteps:

a. -) taking between 1 and 10 g of fermented maize liquor and/or 3 to 30ml of soya protein hydrolysate,

b. -) mixing with a volume of 50 ml of water,

c. -) stirring to complete homogenization, and

d. -) adding sterile water whilst stirring to a final volume of 100 mlof final adhesive composition.

Another object of the present invention is a method for quantifying theadhesion capacity to the surface of vegetable seeds of fluorescentmicrospheres by means of the adhesive composition of the invention wherethe microspheres represent the fungus spores which comprise thefollowing steps:

a. -) taking the adhesive composition of the present invention,

b. -) mixing the adhesive composition of step a) with silica orpolyethylene fluorescent microspheres with a particle size of between1-120 microns and continuing to stir,

c. -) placing the vegetable seeds into contact with the mixture of stepb),

d. -) drying the seeds at room temperature,

e. -) obtaining images of the fluorescent microspheres adhered to theseeds by fluorescence microscopy, exposing them to ultraviolet (UV)light corresponding to a wavelength of 365 nm, and

f. -) counting the fluorescent microspheres adhered to the seeds basedon the images obtained in step e).

According to a preferred aspect, step c) is implemented by submergingthe vegetable seeds in the mixture of step b) in a receptacle in avolumetric ratio (seeds:final volume) of 1:3, continuing to stir themixture for at least 1 minute between 50 and 100 rpm, concluding withdecantation and removing the excess liquid from the seeds.

According to another preferred aspect, step c) is carried out byspraying the mixture of step b) on the vegetable seeds in a homogenousmanner using a proportion of 1 l of mixture per 100 kg of seeds.

According to a preferred aspect, the microspheres have a density of 0.98g/cm³ and a particle size range of 1-120 microns.

According to a preferred aspect, the quantification method is suitablefor cereal seeds.

According to a preferred aspect, the quantification method is suitablefor arbuscular mycorrhizal spores of the genera Rhizophagus intraradicesand Funneliformis mosseae.

Another object of the present invention is the use of the adhesivecomposition of the present invention for catalyzing the adhesion offungus spores to the surface of vegetable seeds.

Another object of the present invention is the use of the adhesivecomposition of the present invention for catalyzing the adhesion ofarbuscular mycorrhizal spores of the genera Rhizophagus intraradices andFunneliformis mosseae.

EXEMPLARY EMBODIMENTS

The following examples are embodiments of the present invention, but arenot limiting of the same.

Example 1. Obtaining the Adhesive Composition of the Present Invention

For the following example, three batches of 3 adhesive compositions havebeen made:

-   -   Sample 1: 15 ml of soya protein hydrolysate and water to        complete a final volume of 100 ml of composition.    -   Sample 2: 4 grams of fermented maize liquor and water to        complete a final volume of 100 ml.    -   Sample 3: Control sample (A) consisting of sterile water.

All the samples were obtained by the following method:

a. -) taking a suitable quantity of fermented maize liquor and/or soyaprotein hydrolysate,

b. -) mixing with a volume of 50 ml of water,

c. -) stirring to complete homogenization, and

d. -) adding sterile water whilst stirring to a final volume of 100 mlof final adhesive composition.

Example 2. Method for Quantifying Fluorescent Microspheres Adhered tothe Surface of the Seeds by Means of an Adhesive Composition Based onSoya Protein Hydrolysate

The following example represents a proof of concept for the efficacy ofthe adhesive composition corresponding to Sample 1 of example 1.

The steps carried out for quantifying by fluorescence were thefollowing:

a. -) taking Sample 1,

b. -) adding 36 mg of fluorescent microspheres and stirring to completehomogenization,

c. -) submerging the seeds to be treated in the mixture of the previousstep, in a bottle with a lid in a volumetric proportion of 1:3 (volumeof seeds:final volume), continuing to stir for at least 1 minute between50 and 100 rpm and decanting the liquid and removing the excess andkeeping the seeds,

d. -) drying the seeds at room temperature,

e. -) obtaining images by fluorescence microscopy, exposing the dryseeds to UV light, and

f. -) counting the microspheres adhered to the seeds based on the imagesobtained in the previous step.

At this point, in order to be able to assess the adhesion capacity, theseeds are placed in a container with a lid with a capacity equal to 300ml and continued to be stirred for 15 minutes between 50 and 100 rpmwith the aim of evaluating the adhesion force of the microspheres on thesurface of the seed. Then, the seeds are removed from the container,they are dried at room temperature and images are again obtained byfluorescence microscopy, exposing the seeds to UV light in order tocount the microspheres adhered to the seeds based on the images obtainedin the previous step and thus to be able to calculate the percentage ofmicrospheres joined to the seed after stirring by means of the followingmathematical formula:

Microspheres of seeds after stirring×100

Microspheres of seeds prior to stirring

Thus the percentage value of the microspheres adhered to the surface ofthe seeds is obtained after an aggressive stirring process.

Example 3. Method for Quantifying Fluorescent Microspheres Adhered tothe Surface of the Seeds by Means of an Adhesive Composition Based onFermented Maize Liquor

The following example represents a proof of concept of the efficacy ofthe adhesive composition corresponding to Sample 2 of example 1.

a. -) taking Sample 2,

b. -) adding 36 mg of fluorescent microspheres and stirring to completehomogenization,

c. -) spraying, by means of the spraying method, the mixture of step b)on the vegetable seeds in a homogenous manner using a proportion of 11of mixture per 100 kg of seeds,

d. -) drying the seeds at room temperature,

e. -) obtaining images by fluorescence microscopy, exposing the dryseeds to UV light, and

f. -) counting the microspheres adhered to the seeds based on the imagesobtained in the previous step.

At this point, in order to be able to assess the adhesion capacity, theseeds are placed in a container with a lid with a capacity equal to 300ml and continued to be stirred for 15 minutes between 50 and 100 rpmwith the aim of evaluating the adhesion force of the microspheres on thesurface of the seed. Then, the seeds are removed from the container andimages are again obtained by fluorescence microscopy, exposing the seedsto UV light in order to count the microspheres adhered to the seedsbased on the images obtained in the previous step and thus to be able tocalculate the percentage of microspheres joined to the seed afterstirring by means of the following mathematical formula

Microspheres of seeds after stirring×100

Microspheres of seeds prior to stirring

Thus the percentage value of the microspheres adhered to the surface ofthe seeds is obtained after an aggressive stirring process.

Example 4. Comparative Example for Assessing the Adhesion Capacity ofthe Adhesive Composition on Cereal Seeds

In the experiments of examples 2 and 3, a total of 300 wheat, maize andsoya seeds were used in each experiment and they were distributed ingroups of 100 seeds per each class. Additionally, a control experimentwas carried out following the same steps of examples 2 and 3, butsubstituting the adhesive composition for sterile water as a manner ofcontrol, that is to say using Sample 3 as the control sample. Eachexperiment was carried out three times.

Table 1 shows the number of fluorescent microspheres adhered to thesurface of the seeds (averages and standard deviations) prior to andafter washing the seeds with aggressive stirring. The percentages ofmicrospheres joined to the surface of the seeds after aggressiveagitation with respect to the number of microspheres joined to thesurface of the seeds after normal agitation are shown in parentheses;that is to say, the percentages for retaining fluorescent microspheresafter aggressive agitation are shown between parentheses.

TABLE 1 Wheat Maize Soya Prior to Prior to Prior to stirring Afterstirring stirring After stirring stirring After stirring A (control)  5.3 ± 0.1^(a) 2.0 ± 0.1 (38) 29.3 ± 1.0 18.9 ± 1.3 (64) 26.3 ± 1.220.7 ± 1.3 (78) Sample 1 13.0 ± 0.3 9.2 ± 0.2 (71) 66.0 ± 3.1 57.5 ± 2.8(87) 67.4 ± 3.7 62.9 ± 3.5 (93) Sample 2 12.5 ± 0.4 9.7 ± 0.3 (78) 65.8± 4.4 57.6 ± 2.6 (87) 68.4 ± 2.9 64.8 ± 3.0 (95)

After carrying out this experiment, it was observed that the adhesioncapacity of the adhesive composition meant that the percentage of seedsmaintaining the fluorescent microspheres adhered after the aggressivestirring process was significantly higher than in the control samples.

The invention claimed is:
 1. A method for quantifying the adhesioncapacity of fluorescent microspheres to the surface of vegetable seedsby means of an adhesive composition, characterized in that it comprisesthe following steps: a) taking an adhesive composition comprising anaqueous solution of corn steep liquor and/or soya protein hydrolysates,b) mixing the adhesive composition of step a) with silica orpolyethylene fluorescent microspheres with a particle size of between1-120 microns and continuing to stir, c) placing the vegetable seedsinto contact with the mixture of step b), d) drying the seeds at roomtemperature, e) obtaining images of the fluorescent microspheres adheredto the seeds by fluorescence microscopy, exposing them to ultraviolet(UV) light corresponding to a wavelength of 365 nm, and f) counting thefluorescent microspheres adhered to the seeds based on the imagesobtained in step e).
 2. The method according to claim 1, wherein theadhesive composition comprises from 3 to 30 ml of soya proteinhydrolysate and/or 1 to 10 g of corn steep liquor in water to complete afinal volume of 100 ml.
 3. The method according to claim 1 wherein stepc) is implemented by submerging the vegetable seeds in the mixture ofstep b) in a receptacle in a volumetric ratio (seeds:final volume) of1:3, continuing to stir the mixture for at least 1 minute between 50 and100 rpm, concluding with decantation and removing the excess liquid fromthe seeds.
 4. The method according to claim 1, wherein in that step c)is carried out by spraying the mixture of step b) on the vegetable seedsin a homogenous manner using a proportion of 11 of mixture per 100 kg ofseeds.
 5. The method according to claim 1, wherein the microspheres havea density of 0.98 g/cm³ and a particle size range of 1-120 microns. 6.The method according to claim 1, wherein said vegetable seeds are cerealseeds.
 7. A method for catalyzing the adhesion of fungus sporescharacterized in that it comprises the addition of an adhesivecomposition comprising an aqueous solution of corn steep liquor and/orsoya protein hydrolysates to the surface of vegetable seeds.
 8. Themethod according to claim 7, wherein said adhesive composition comprisesfrom 3 to 30 ml of soya protein hydrolysate and/or 1 to 10 g of cornsteep liquor in water to complete a final volume of 100 ml.
 9. Themethod according to claim 7, wherein said fungus spores are arbuscularmycorrhizae spores.
 10. The method according to claim 7, wherein saidarbuscular mycorrhizae spores are of the genera Rhizophagus intraradicesand Funneliformis mosseae.